Heredity (2005) 94, 159–165 & 2005 Nature Publishing Group All rights reserved 0018-067X/05 $30.00 www.nature.com/hdy Inbreeding depression in self-incompatible and self-compatible populations of Leavenworthia alabamica JW Busch Department of Biology, Indiana University, 1001 E 3rd St Bloomington, IN, USA Inbreeding depression is one of the leading factors prevent- self-fertilization led to the production of fewer and smaller ing the evolution of self-fertilization in plants. In populations seeds within self-incompatible populations. Moreover, in- where self-fertilization evolves, theory suggests that natural breeding depression was observed in eight of 11 offspring selection against partially recessive deleterious alleles will traits within self-incompatible populations of L. alabamica.In reduce inbreeding depression. The purpose of this study was contrast, there was significant inbreeding depression only in to evaluate this hypothesis by comparing the magnitude of flower number within self-compatible populations. The inbreeding depression in self-incompatible and self-compa- results of this study are consistent with the idea that self- tible populations of Leavenworthia alabamica. Within-popu- fertilization selectively removes partially recessive deleter- lation crosses were conducted to compare the quantity and ious alleles causing inbreeding depression in natural plant quality of offspring produced by outcrossing and self- populations. However, in plant species such as L. alabamica fertilization. These progeny were grown in a common where self-compatibility may evolve in small populations greenhouse and inbreeding depression was measured in following long-distance dispersal, declines in inbreeding germination, survival, biomass, transition rate to flowering, depression may also be facilitated by genetic drift. flower number, petal length, pollen grains/anther, pollen Heredity (2005) 94, 159–165. doi:10.1038/sj.hdy.6800584 viability, and ovule number. In comparison to outcrossing, Publishedonline24November2004 Keywords: deleterious mutations; genetic drift; genetic load; purging; self-fertilization Introduction equilibrium frequency of lethal and sublethal mutations that are nearly recessive because these alleles are Self-fertilization has an innate transmission advantage exposed to natural selection in the homozygous state that should lead to the elimination of cross-fertilization (Lande and Schemske, 1985; Charlesworth et al, 1990; because selfing individuals pass on an extra copy of their Byers and Waller, 1999). This hypothesis has been tested chromosomes through seeds (Fisher, 1941). However, the by forcibly self-fertilizing naturally outcrossing families initial spread of genes for self-fertilization can be halted of plants for several generations (Barrett and by inbreeding depression, or the lowered viability and Charlesworth, 1990; Carr and Dudash, 1997; Dudash fecundity of selfed plants (Lloyd, 1979; Lande et al, 1997; Willis, 1999) or by comparing populations or and Schemske, 1985; Charlesworth et al, 1990; Jarne and lineages of plants that have divergent rates of self- Charlesworth, 1993). Genetic models have explored the fertilization in natural populations (Holtsford and conditions favoring the spread of genes for self-fertiliza- Ellstrand, 1990; Carr and Dudash, 1996; Johnston and tion in cross-fertilizing populations. In general, rates of Schoen, 1996; Fishman, 2001; Takebayashi and Delph, self-fertilization will increase in populations whenever 2001). Results of these studies suggest that inbreeding selfed plants are at least half as fit as outcrossed plants, if depression may be reduced by self-fertilization, although genetic associations between fitness loci and selfing-rate purging may not always occur in response to periods of modifier loci are not considered (Lande and Schemske, inbreeding (Byers and Waller, 1999). 1985; Campbell, 1986; Charlesworth et al, 1990; Uyeno- Leavenworthia alabamica is an ideal species in which to yama and Waller, 1991a, b; Lloyd, 1992). test the idea that self-fertilization reduces inbreeding Evidence suggests that inbreeding depression is depression in natural plant populations (Lloyd, 1965). In caused mainly by the expression of partially recessive this species, there is variation among populations in the deleterious alleles in the homozygous state (Dudash and prevalence of self-incompatibility. In particular, self- Carr, 1998; Charlesworth and Charlesworth, 1999; Willis, incompatibility predominates at the center of the species 1999). The evolution of self-fertilization should lower the range and self-compatibility occurs in smaller popula- tions at the border of the species range (Lloyd, 1965). Individuals within self-compatible populations produce Correspondence: JW Busch, Department of Biology, Indiana University, smaller flowers, have lower pollen to ovule ratios, and 1001 E. 3rd St Bloomington, IN 47405, USA. E-mail: [email protected] produce a greater proportion of fruit through autono- Received 2 January 2004; accepted 15 July 2004; published online 24 mous self-fertilization (Lloyd, 1965; Busch, unpublished November 2004 data). There is likely to have been a history of natural Inbreeding depression in Leavenworthia JW Busch 160 selection in these populations to circumvent the ancestral incubator with 14 h days (151C) and 10 h nights (121C), sporophytic self-incompatibility reaction found through- and were moistened daily with 3 ml of autoclaved water. out the Brassicaceae and the genus Leavenworthia (Lloyd, These plants were then grown in a common greenhouse 1967; Bateman, 1955). The purpose of this experiment to minimize any potential environmental maternal and was to test the hypothesis that the evolution of self- paternal effects that would influence the offspring compatibility in L. alabamica is associated with reductions performance. Seedlings were transplanted into 3 in. pots in the genetic load causing inbreeding depression. containing a 1:1 ratio of MetroMix (Scotts-Sierra Horti- cultural Products, Marysville, OH, USA) and sterilized soil following the emergence of both cotyledons. Plants Methods received 14 h of artificial light in the Indiana University greenhouse to promote flowering. Study system L. alabamica Rollins (Alabama glade cress; Brassicaceae) is a winter annual endemic to the limestone cedar glades Crossing design and greenhouse experiment of the Moulton Valley in northern Alabama (Rollins, Plants were used as parents in a crossing design to 1963). These cedar glades are typified by exposed and quantify the fitness effects of outcrossing and selfing. slowly eroding beds of limestone that are covered with a Outcrossed and selfed offspring were generated by thin and moist layer of soil (Baskin et al, 1995). Seed outcrossing and forcibly self-fertilizing plants in all germination occurs in the late fall following a long populations, respectively. Outcrossed offspring were period of summer desiccation required to break dor- produced by using randomly chosen pollen donors from mancy. Flowering occurs from early March until the within populations. To successfully produce selfed off- middle of April. Seeds mature within siliques on spring, it was necessary to circumvent the self-incompat- maternal plants in the early summer. There is likely to ibility system active in some populations through bud- be limited pollen dispersal between populations because pollination. By forcing styles to accept pollen before of the short flight distances of native pollinators (Andrena flowers open, bud-pollinations allow self pollen tubes to spp. and Halictus ligatus) and the relatively large evade detection by maternal style proteins that normally distances between cedar glades. Seed dispersal is greatly inhibit the germination of related pollen grains after limited because seeds are passively dispersed following flowers have opened (Bateman, 1955). Bud-pollinations maturation. Nevertheless, the most likely form of gene were used in all crosses to ensure that flower damage flow between populations is thought to occur following would not be responsible for the reduced performance of the dispersal of seeds by rare flooding events (Lloyd, self-fertilized offspring (Cabin et al, 1996). Overall, a total 1965). of 435 families were generated by outcrossing and selfing. Each morning, flowers with visible petal emargina- Seed collection and growth of parental plants tions were chosen for bud-pollinations. All of the sepals, Attempts were made during the spring of 2002 to locate petals, and anthers were then removed with forceps from the naturally occurring populations described by Lloyd the bud of the ovule parent. Pollen parents were selected (1965). Populations with high rates of autonomous self- haphazardly, with the restriction that each plant was fertilization, small petal size, and low pollen number used as a pollen donor no more than three times. should have long histories of inbreeding (Byers and Following pollen transfer, the date of bud-pollination Waller, 1999). In this study, I collected seed from five and the identity of the pollen donor were recorded for large, cross-fertilizing populations: Hatton (34.509931N, each flower. Marked fruits were allowed to mature for 1 87.442041W), Isbell (34.457251N, 87.752981W), Newburg month and were then collected for storage. These seeds (34.470491N, 87.573001W), Tharptown (34.593271N, were put in envelopes, placed in a 301C oven for 1 week, 87.571981W), Waco (34.478661N, 87.627461W) and five small, and then stored at 221C for 3 months to